High-confidence placement of difficult-to-fit fragments into electron density by using anomalous signals - a case study using hits targeting SARS-CoV-2 non-structural protein 1 DOI Open Access
Shumeng Ma,

Vitaliy Mykhaylyk,

Matthew W. Bowler

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: June 16, 2023

Abstract The identification of multiple simultaneous orientations small molecule inhibitors binding to a protein target is common challenge. It has recently been reported that the conformational heterogeneity ligands widely underreported in Protein Data Bank, which likely impede optimal exploitation improve affinity these 1 . Significantly less even known about for fragments (< 300 Da) although this information would be essential subsequent fragment optimisation using growing, linking or merging and rational structure-based design. Here we use hits SARS-CoV-2 non-structural (nsp1) N-terminal domain propose general procedure unambiguously identifying 2-dimensional containing either sulphur chloro substituents within wavelength range most tunable beamlines. By measuring datasets at two energies, tuneable beamline operating vacuum optimised data collection very low X-ray show anomalous signal can used identify and/or verify conformations. Although specific case identified positions chlorine bound their target, are confident work further expanded additional atoms ions often occur fragments. Finally, our improvements understanding will also serve advance nsp1 targeting hits.

Language: Английский

SARS-CoV-2 proteins structural studies using synchrotron radiation DOI
Maksim N. Kosenko, G. S. Onkhonova, Ivan M. Susloparov

et al.

Biophysical Reviews, Journal Year: 2023, Volume and Issue: 15(5), P. 1185 - 1194

Published: Sept. 29, 2023

Language: Английский

Citations

2
Insights into the evolution of mutations in SARS-CoV-2 non-spike proteins DOI

Rimanshee Arya,

Preeti Tripathi, Karthik Nayak

et al.

Microbial Pathogenesis, Journal Year: 2023, Volume and Issue: 185, P. 106460 - 106460

Published: Nov. 21, 2023

Language: Английский

Citations

2
Protein structure-based approaches against COVID-19 DOI Creative Commons

Ta-Chou Huang,

Kung–Hao Liang,

Tai-Jay Chang

et al.

Journal of the Chinese Medical Association, Journal Year: 2023, Volume and Issue: 87(2), P. 139 - 141

Published: Dec. 20, 2023

The coronavirus disease 2019 (COVID-19) pandemic has had a major impact on human life. This review highlights the versatile roles of both classical and modern structure-based approaches for COVID-19. X-ray crystallography, nuclear magnetic resonance spectroscopy, cryogenic electron microscopy are three cornerstones structural biology. These technologies have helped provide fundamental detailed knowledge regarding severe acute respiratory syndrome 2 (SARS-CoV-2) related host proteins as well enabled identification its target sites, facilitating cessation transmission. Further progress into protein structure modeling was made using derived from homology integrated with artificial intelligence (AI), advanced computational simulation tools to actively guide design new vaccines development anti-SARS-CoV-2 drugs. presents practical contributions future directions

Language: Английский

Citations

2
Structure–function mapping and mechanistic insights on the SARS CoV2 Nsp1 DOI
Bruno A. Salgueiro, Margarida Saramago, Mark D. Tully

et al.

Protein Science, Journal Year: 2024, Volume and Issue: 33(12)

Published: Nov. 25, 2024

Abstract Non‐structural protein 1 (Nsp1) is a key component of the infectious process caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV2), responsible for COVID‐19 pandemic. Our previous data demonstrated that Nsp1 can degrade both RNA and DNA in absence ribosome, dependent on metal ions Mn 2+ , Ca or Mg (Salgueiro et al., SARS‐CoV2 metal‐dependent endonuclease. Biometals. 2024;37:1127–1146). The composed two structural domains: N‐terminal domain (NTD) C‐terminal (CTD), connected loop. To elucidate function each domain, we generated four truncated versions containing either NTD CTD. results indicate domains play distinct functional roles. Specifically, involved nucleotide binding regulation, while CTD acts as catalytic domain. Moreover, tyrosyl radical was detected during nuclease activity, an in‐depth analysis different constructs suggested Y136 could be this process. Indeed, our show Y136F variant lacks activity but retains its activity. Furthermore, observed has propensity to associate with hydrophobic environments, suggesting it might cell membranes. However, cellular association requires further investigation. This study sheds light functions individual Nsp1, providing valuable insights into mechanism action Coronaviruses.

Language: Английский

Citations

0
Structure-based Search for novel c-di-AMP Synthase Inhibiting Fragments DOI Creative Commons
Tim Benedict Garbers

Published: Jan. 1, 2023

One of the major threats for global health system is increasing number antibiotic resistant bacterial strains. The misuse antibiotics during Covid pandemic aggravates these problems. Therefore, development new mandatory to fight rising threat multi-resistant bacteria. essential second messenger c-di-AMP was discovered in 2008 and mainly found gram positive It has been identified several human pathogens like Listeria monocytogenes, Staphylococcus aureus or Enterococcus faecalis. a key player regulation pathways DNA integrity scanning, cell wall metabolism osmolyte homeostasis. Five different protein classes are able synthesize They all have diadenylate cyclase domain (DAC) common need dimerize produce metal-ion dependent manner from two ATP molecules. class CdaA consists three N-terminal transmembrane helices followed by DAC often sole pathogenic bacteria aureus, Streptococcus pneumoniae faecium. essentiality renders as potential target novel substances. goal this thesis establishment crystallization suitable fragment screening campaign order identify starting points inhibitors CdaA. first part work focused on soil bacterium Bacillus subtilis To obtain constructs well suited crystallography, CdaAs organisms were N- C-terminal truncated solely consist domain. purity homogeneity every purified verified via SDS-PAGE DLS. prove enzymatic functionality proteins, coralyne activity assay applied with varying divalent cations. All exhibit presence manganese ions while their magnesium- cobalt-ions differs. explore variations, trials favorable carried out. As result, crystal structure faecium complex magnesium solved at resolution 2.1 Å (EfCdaA) 2.2 (SpCdaA). Unfortunately, both models represent post catalytic state where metal ion not coordinated catalytically active way, giving no further insights into structural basis differing specificity. Nevertheless, if obtained structures compared monocytogenes c-di-AMPa conserved interaction pattern could be observed, rendering interacting amino acids possible targets inhibitor design. In fragments which can reduce CdaA, previously mentioned APO state. For successful. However, does matches requirements crystallographic crystals diffracted just 2.4 required months grow. contrast, up 1.6 highly reproduceable. Further optimization condition led greatly campaign. Through following using F2X-Entry screen, 32 unique interact BsCdaA. Mapping Consurf model reduced six bound active/dimerization site. These also biochemically characterized malachite green assay. none BsCdaA, an alternative approach out inhibitory compound. Here, BsCdaA AMP, determined served point identification By computational defragmentation employing SeeSar, adenine AMP largely responsible biding capability AMP. Combing methods computer aided drug design information gained X-ray Janus kinase Ruxolitinib compound binding mode towards proven exhibits same it concluded binds site protein, most likely acts competitive Moreover, IC50 value 2.7 µM Determining show activity, underlining conservation protein-ligand interaction. Besides vitro studies, vivo experiments utilizing strains results suggest high specificity vivo.

Language: Английский

Citations

0
An evolutionarily conserved strategy for ribosome binding and inhibition by β-coronavirus non-structural protein 1 DOI Creative Commons

Stephanie F. Maurina,

John P. O’Sullivan,

Geetika Sharma

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: June 8, 2023

An important pathogenicity factor of SARS-CoV-2 and related coronaviruses is Nsp1, which suppresses host gene expression stunts antiviral signaling. Nsp1 binds the ribosome to inhibit translation through mRNA displacement induces degradation mRNAs an unknown mechanism. Here we show that Nsp1-dependent shutoff conserved in diverse coronaviruses, but only from β-CoV inhibits binding. The C-terminal domain all Nsp1s confers high-affinity ribosome-binding despite low sequence conservation. Modeling interactions four identified few absolutely amino acids that, together with overall conservation surface charge, form domain. Contrary previous models, inefficient inhibitor. Instead, Nsp1-CTD likely functions by recruiting Nsp1's N-terminal "effector" Finally, a viral cis -acting RNA element has co-evolved fine-tune function, does not provide similar protection against viruses. Together, our work provides new insight into diversity ribosome-dependent host-shutoff knowledge could aide future efforts pharmacological targeting SARS-CoV-2, also human-pathogenic β-coronaviruses. Our study exemplifies how comparing highly divergent variants can help dissect different modalities this multi-functional protein.

Language: Английский

Citations

0
High-confidence placement of difficult-to-fit fragments into electron density by using anomalous signals - a case study using hits targeting SARS-CoV-2 non-structural protein 1 DOI Open Access
Shumeng Ma,

Vitaliy Mykhaylyk,

Matthew W. Bowler

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: June 16, 2023

Abstract The identification of multiple simultaneous orientations small molecule inhibitors binding to a protein target is common challenge. It has recently been reported that the conformational heterogeneity ligands widely underreported in Protein Data Bank, which likely impede optimal exploitation improve affinity these 1 . Significantly less even known about for fragments (< 300 Da) although this information would be essential subsequent fragment optimisation using growing, linking or merging and rational structure-based design. Here we use hits SARS-CoV-2 non-structural (nsp1) N-terminal domain propose general procedure unambiguously identifying 2-dimensional containing either sulphur chloro substituents within wavelength range most tunable beamlines. By measuring datasets at two energies, tuneable beamline operating vacuum optimised data collection very low X-ray show anomalous signal can used identify and/or verify conformations. Although specific case identified positions chlorine bound their target, are confident work further expanded additional atoms ions often occur fragments. Finally, our improvements understanding will also serve advance nsp1 targeting hits.

Language: Английский

Citations

0